Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins. However, most gram-positive bacteria, including many strict aerobes, do not produce glutathione. Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.
Actinomycetes, including Streptomyces and Mycobacterium, do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1D-myo-inosityl 2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins). The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione. However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in several enzymatic processes. First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.) pp 517-551, John Wiley & Sons, et al.). An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al. (1997) FEBS Lett. 409:221-222). This enzyme has been sequenced (A. Norin, et al. (1997) Eur. J. Biochem. 248:282-289).
The second enzymatic process involves a mycothiol homolog of glutathione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al. (1999) J. Amer. Chem. Soc. 120:11538-11539; M. P. Patel et al. (1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126). The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.
A general mycothiol-dependent detoxification process has been described in M. smegmatis in which MSH forms S-conjugates (MSR) with reactive electrophiles, including some antibiotics, and MSR is subsequently degraded by the enzyme mycothiol S-conjugate amidase to produce GlcN-Ins and AcCySR, a mercapturic acid, which is excreted from the cell; in MSR R is derived from the electrophile (Newton, et al. (2000) Biochemistry 39:10739-10746).
The biosynthesis of MSH has been identified as involving four steps: (1) formation of GlcNAc-Ins; (2) deacetylation of GIcNAc-Ins to produce GleN-Ins; (3) ligation of GleN-Ins to Cys to produce Cys-GlcN-Ins; (4) acetylation of Cys-GIcN-Ins by acetyl-CoA to produce MSH (Bornemann, et al. (1997) Biochem. J. 325:623-629; Anderberg, et al. (1998) J. Biol. Chem. 273:30391-7; Newton, et al. (2000) J. Bacteriol. 182:6958-6963). The genes encoding these biosynthesis steps have been designated mshA, mshB, mshC, and mshD but the only biosynthetic gene identified thus far is the mshB gene encoding the deacetylase MshB (Newton, et al. (2000) J. Bacteriol. 182:6958-6963).
The structure of mycothiol, 1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (AcCys-GlcN-Ins), makes it resistant to heavy-metal-catalyzed autoxidation (Newton et al. 1995) and it appears to have functions analogous to those of glutathione. A mycothiol-dependent formaldehyde dehydrogenase has been identified (Misset-Smits et al. 1997; Norin et al. 1997). Mycobacterium smegmatis mutants defective in MSH biosynthesis exhibit enhanced sensitivity to hydrogen peroxide and modified sensitivity to antibiotics (Newton et al. 1999). Alkylating agents are detoxified by mycothiol and the resulting S-conjugates cleaved by an amidase to produce the N-acetylcysteine derivative (mercapturic acid), which is excreted from the cell (Newton et al. 2000b). A mycothiol disulfide reductase maintains mycothiol in the reduced state (Patel and Blanchard 1999; Patel and Blanchard 2001).
Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione and for identification of as yet unidentified biosynthesis genes.
Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases. Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals.